Abstract
Epstein Barr Virus (EBV) replicates in oral epithelial cells and gains entry to B-lymphocytes. In B-lymphocytes, EBV expresses a restricted subset of genes, the Latency III program, which converts B-lymphocytes to proliferating lymphoblasts. Latent Membrane Protein 1 (LMP1) and the other Latency III associated proteins are also expressed during virus replication. LMP1 is essential for virus replication and egress from Akata Burkitt Lymphoma cells, but a role in epithelial cell replication has not been established. Therefore, we have investigated whether LMP1 enhances EBV replication and egress from HEK293 cells, a model epithelial cell line used for EBV recombinant molecular genetics. We compared wild type (wt) and LMP1-deleted (LMP1Δ) EBV bacterial artificial chromosome (BAC) based virus replication and egress from HEK293. Following EBV immediate early Zta protein induction of EBV replication in HEK293 cells, similar levels of EBV proteins were expressed in wt- and LMP1Δ-infected HEK293 cells. LMP1 deletion did not impair EBV replication associated DNA replication, DNA encapsidation, or mature virus release. Indeed, virus from LMP1Δ-infected HEK293 cells was as infectious as EBV from wt EBV infected HEK cells. Trans-complementation with LMP1 reduced Rta expression and subsequent virus production. These data indicate that LMP1 is not required for EBV replication and egress from HEK293 cells.
Highlights
Epstein-Barr Virus (EBV), like most human herpes viruses, spreads through saliva and is believed to initally replicate in the oropharyngeal epithelium
Construction of an Latent Membrane Protein 1 (LMP1)-deleted EBV bacterial artificial chromosome (BAC), LMP1D BAC The LMP1 ORF was replaced with the chloramphenicol acetyl transferase (CAT) gene flanked by FLP recombinase target (FRT) sites, using lambda red mediated homologous recombination and wt EBV-BAC (MD1 [23]) (LMP1DCAT, Fig. 1A)
Wt and LMP1D BAC HEK293 cell clones were isolated by selection for puromycin resistance, which is expressed from the BAC F plasmid
Summary
Epstein-Barr Virus (EBV), like most human herpes viruses, spreads through saliva and is believed to initally replicate in the oropharyngeal epithelium (for review see [1]). Circulating naıve B cells are infected early in primary human infection. In these cells, a latency III (LTIII) program of virus gene expression promotes infected B cell replication, and differentiation into memory B cells, which are the reservoir for reactivated EBV replication and infection of neighboring epithielial cells [2,3] [4,5]. Because EBV LTIII infection transforms B lymphocytes into immortal lymphoblast cell lines (LCLs), in vitro, the genetics and biochemistry of LTIII infection have been extensively studied. Latent Membrane Protein 1 (LMP1) induces NFkB, has Ras-like transforming effects on cells, and is expressed in most EBV-related malignancies including Post-transplant lymphoproliferative disorders, Hodgkin’s Disease, and Nasopharyngeal Carcinoma (for review see [1]). LMP1-mediated NFkB activation is required for B cell growth and survival [6,7,8,9,10]
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